This study aims to investigate the expression and significance of GOLPH3 in human gastric cancer progression and prognosis. Using immunohistochemistry (IHC) and real-time reverse transcriptase polymerase chain reaction assay, we identified abnormally elevated expression of GOLPH3 in gastric cancer tissues compared to paired normal stomach mucosa tissues in 40 patients. In addition, the enzyme-linked immunosorbent assay (ELISA) was used to quantify serum GOLPH3 concentrations in the same 40 gastric cancer patients and 40 healthy individuals. ELISA revealed significantly higher serum concentrations of GOLPH3 in gastric cancer patients compared to healthy individuals (p = 0.002). In order to investigate the correlations between GOLPH3 and the clinicopathological features of gastric cancer, the expression of GOLPH3 in 123 gastric cancer patients were detected by IHC, and the results showed that overexpression of GOLPH3 was associated with the size of the tumor (p = 0.013), histological grade (p = 0.002), depth of invasion (p < 0.001), lymph node metastasis (p < 0.001), distant metastasis (p = 0.018), and TNM stage (p < 0.001). Kaplan-Meier survival analysis showed that high GOLPH3 expression exhibited a significant correlation with poor prognosis for gastric cancer patients. Further, Cox multivariate analysis indicated that GOLPH3 expression level was an independent prognostic factor for patients after radical resection. In conclusion, the overexpression of GOLPH3 is closely related to the progression of gastric cancer and might be regarded as an independent predictor of poor prognosis for gastric cancer.